Probes for INS

Peripheral nerve injuries are accompanied by inflammatory reactions, over-activation of which may hinder recovery. Among pro-inflammatory pathways, inflammasomes are one of the most potent, leading to release of active IL-1β. Our aim was to understand how inflammasomes participate in central inflammatory reactions accompanying peripheral nerve injury.After axotomy of the sciatic nerve, priming and activation of the NLRP3 inflammasome was examined in cells of the spinal cord. Regeneration of the nerve was evaluated after coaptation using sciatic functional index measurements and retrograde tracing.In the first 3 days after the injury, elements of the NLRP3 inflammasome were markedly upregulated in the L4-L5 segments of the spinal cord, followed by assembly of the inflammasome and secretion of active IL-1β. Although glial cells are traditionally viewed as initiators of neuroinflammation, in this acute phase of inflammation, inflammasome activation was found exclusively in affected motoneurons of the ventral horn in our model. This process was significantly inhibited by 5-BDBD, a P2X4 receptor inhibitor and MCC950, a potent NLRP3 inhibitor. Although at later time points the NLRP3 protein was upregulated in microglia too, no signs of inflammasome activation were detected in these cells. Inhibition of inflammasome activation in motoneurons in the first days after nerve injury hindered development of microgliosis in the spinal cord. Moreover, P2X4 or inflammasome inhibition in the acute phase significantly enhanced nerve regeneration on both the morphological and the functional levels.Our results indicate that the central reaction initiated by sciatic nerve injury starts with inflammasome activation in motoneurons of the ventral horn, which triggers a complex inflammatory reaction and activation of microglia. Inhibition of neuronal inflammasome activation not only leads to a significant reduction of microgliosis, but has a beneficial effect on the recovery as well.

Central nervous system (CNS)-mediated symptoms, such as fatigue, depression, and hyperalgesia, are common complications among patients with rheumatoid arthritis (RA). However, it remains unclear how the peripheral pathology of RA spreads to the brain. Accumulated evidence showing an association between serum cytokine levels and aberrant CNS function suggests that humoral factors participate in this mechanism. In contrast to the well-known early responses of microglia (CNS-resident immune cells) in the area postrema [AP; a brain region lacking a blood-brain barrier (BBB)] to experimental inflammation, microglial alterations in the AP during chronic inflammation like RA remain unclear. Therefore, to determine whether microglia in the AP can react to persistent autoimmune-arthritis conditions, we analyzed these cells in a mouse model of collagen-induced arthritis (CIA).Microglial number and morphology were analyzed in the AP of CIA and control mice (administered Freund's adjuvant or saline). Immunostaining for ionized calcium-binding adaptor molecule-1 was performed at various disease phases: "pre-onset" [post-immunization day (PID) 21], "establishment" (PID 35), and "chronic" (PID 56 and 84). Quantitative analyses of microglial number and morphology were performed, with principal component analysis used to classify microglia. Interleukin-1β (IL-1β) mRNA expression was analyzed by multiple fluorescent in situ hybridization and real-time polymerase chain reaction. Behavioral changes were assessed by sucrose preference test.Microglia in the AP significantly increased in density and exhibited changes in morphology during the establishment and chronic phases, but not the pre-onset phase. Non-subjective clustering classification of cell morphology (CIA, 1,256 cells; saline, 852 cells) showed that the proportion of highly activated microglia increased in the CIA group during establishment and chronic phases. Moreover, the density of IL-1β-positive microglia, a hallmark of functional activation, was increased in the AP. Sucrose preferences in CIA mice negatively correlated with IL-1β expression in brain regions containing the AP.Our findings demonstrate that microglia in the AP can sustain their activated state during persistent autoimmune arthritis, which suggests that chronic inflammation, such as RA, may affect microglia in brain regions lacking a BBB and have various neural consequences.

Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune cell-mediated cytokine storm remain unknown. Using a vascularized lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response, which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2-mediated endothelial cell damage thus occurs independently of cytokine storm.

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients.

Astroviruses cause a spectrum of diseases spanning asymptomatic infections to severe diarrhea, but little is understood about their pathogenesis. We previously determined that small intestinal goblet cells were the main cell type infected by murine astrovirus-1. Here, we focused on the host immune response to infection and inadvertently discovered a role for indoleamine 2,3-dioxygenase 1 (Ido1), a host tryptophan catabolizing enzyme, in the cellular tropism of murine and human astroviruses. We identified that Ido1 expression was highly enriched among infected goblet cells, and spatially corresponded to the zonation of infection. Because Ido1 can act as a negative regulator of inflammation, we hypothesized it could dampen host antiviral responses. Despite robust interferon signaling in goblet cells, as well as tuft cell and enterocyte bystanders, we observed delayed cytokine induction and suppressed levels of fecal lipocalin-2. Although we found Ido-/- animals were more resistant to infection, this was not associated with fewer goblet cells nor could it be rescued by knocking out interferon responses, suggesting that IDO1 instead regulates cell permissivity. We characterized IDO1-/- Caco-2 cells and observed significantly reduced human astrovirus-1 infection. Together this study highlights a role for Ido1 in astrovirus infection and epithelial cell maturation.

Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.